The replication of HIV-1 occurs through the reverse-transcription of its RNA genome into DMA. This complex process is chaperoned by the nucleocapsid protein (NC), and occurs through a series of intermediate steps. One sub-step in the replication is minus-strand transfer, in which NC assists the annealing of transactivation response (TAR) DNA to the complementary 5' end of the viral RNA in preparation for the completion of minus-strand synthesis. This step in the reverse transcription is in itself complex, because it involves alternate pathways and intermediates, and requires the presence of NC to achieve the thermodynamically optimal product: fully annealed DNA/RNA. Single molecule spectroscopy (SMS) is an ideal technique to study such an innately heterogeneous thermodynamic process. I propose to use SMS to characterize the pathway of this step in the reverse-transcription process, and to elucidate any crucial intermediates in the annealing of TAR DNA/RNA. Confocal, wide-field, and correlation techniques will be used to study native and mutated nucleic acid/NC interactions. Specifically, the experiments I have proposed are designed to determine the.rate-limiting step in the annealing process, and the nature of the nucleation event.